Ninhydrin

Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is evolved. Ninhydrin is most commonly used to detect fingerprints, as amines left over from peptides and proteins (terminal amines or lysine residues) sloughed off in fingerprints react with ninhydrin.^[1]

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Uses

Ninhydrin can also be used to monitor deprotection in solid phase peptide synthesis (Kaiser Test).^[2] When the growing peptide chain is deprotected, a ninhydrin test yields blue. If the next peptide residue is coupled then the test is colorless or yellow.

Ninhydrin is also used in amino acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with ninhydrin except Proline; Also, certain amino acid chains are degraded. Therefore, separate analysis is required for identifying such amino acids that either react differently or don't react at all with ninhydrin. The rest of the amino acids are then quantified colorimetrically after separation by chromatography.

A solution suspected of containing the ammonium ion can be tested by ninhydrin by dotting it onto a solid support (such as silica gel); treatment with ninhydrin should result in a dramatic purple color if the solution contains this species. In the analysis of a chemical reaction by TLC, the reagent can also be used. It will detect, on the TLC plate, virtually all amines, carbamates and also, after vigorous heating, amides.

Mechanism to generate the ninhydrin chromophore

Note that in order to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yellow-orange in color. for further information see: http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-3-3.html

See also

• Paper chromatography

References

1. ^ http://www.bergen.org/EST/Year5/fingerprint.htm
2. ^ E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Analytical Biochemistry 34 595 (1970)